Omega 3 fatty acids are a family of polyunsaturated fatty acids whose common feature is that the last double bond is located in the third C—C bond starting from the final methyl group of the fatty acid. Omega 3 fatty acids are essential, that is, the human body cannot produce them internally and therefore it is necessary to take them through the diet or through compositions. Due to their polyunsaturated nature, omega 3 fatty acids have very particular physicochemical functions in the human body (ie. very low melting point) and therefore they have been widely studied. Today, it is known that there are up to 10 omega 3 fatty acids (ie. stearic acid), although their presence in the human body is in very small amounts and their physiological activity is very low or absent, except for DHA and/or EPA.
The acid 5,8,11,14,17-eicosapentaenoic or EPA, as well as 4,7,11,13,16,19-docosahexaenoic or DHA are the omega 3 fatty acids with most physiological functions, specially DHA, which has specific functions in retina, sperm, neural tissue etc. Both DHA and EPA have common physiological functions, although DHA has specific physiological functions that no other fatty acid has, particularly in photoreceptors, neural tissue and sperm amongst others. The intake of high doses of DHA increases the levels of EPA, although the reverse does not happen; in addition, DHA does not alter the synthesis of other fatty acids (Voss et al., 1992).
From the physiological perspective, DHA is the most interesting omega 3 fatty acid from the biological viewpoint for human consumption. Since DHA and EPA are present in the same food sources and since EPA is more abundant, initially EPA has been the one that has called more attention, being easier to obtain. However, knowledge on these two fatty acids over the last 15 years has significantly increased the interest for DHA and for its necessary purification since, aside from exceptions, at the most it is present in no more than 10-15% of the fats in the most abundant food sources.
The majority of sources for obtaining omega 3 fatty acids rich in DHA are of marine origin: microalgae (ie. Schizochytrium sp., Crypthecodinium sp., Ulkenia sp., Euglena sp.), crustaceans (ie. krill Euphausia superba), fatty fish (ie. Thunnus ibynnus thynnus or red tunna), and marine mammals; in addition to mushrooms and yeast (ie. Yarrowia lipolytica) and bacterias (ie. Lactobacillus spp.).
The most abundant source with the highest purity and most adequate for obtaining omega 3 fatty acids rich in DHA for human consumption is fish, since the production is of 140 million Tons of fish and shellfish per year (FAO, 2007) and it is estimated that the production of fish oil accounts for 1 million metric tonnes yearly (IFFO, International Fishmeal & Fish oil Organisation). The most suitable fish is tunna and other species with the highest content of Omega 3, being the percentage of DHA beyond 20% in weight; besides, since they are food products they exhibit the highest guarantee and safety in public health. On the other hand, together with fish, krill represents the largest reserve and biomass of DHA of the planet. However, the exploitation of krill to obtain omega 3 fatty acids represents a serious and known risk for trophic chains and the development of species and fish necessary for the human diet. A possibility in the future would be obtaining such krill in krill farms, where there would be no risk for the trophic chains and in addition, they could reproduce in an environment free from contaminants that would normally exist in marine waters.
Numerous authors consider that DHA is a deficient nutrient in most diets worldwide. But, in addition to its relevance as a treatment in DHA deficient diseases, its physiological action makes it into the most relevant cofactor for the treatment and prevention of neurodegenerative disease such as Alzheimer or schizophrenia; retina degenerative diseases, cancer, autoimmune diseases, chronic articular and dermatologic inflammatory diseases, renal and urologic diseases (prostate), androgenic alopecia, alterations in male and female fertility, primary attention disorder or hyperactivity, intellectual and cognitive development, as well as necessary for the visual development and particularly of the macular region, cardiovascular diseases, diabetes and hypertriglyceridemia.
For the treatment of numerous pathologies, it is necessary to use doses tens of times higher to those obtained just with the diet (generally >2-4 grams), which makes it necessary to obtain DHA compositions with high purities in order to achieve the consumption of adequate doses (ie. to obtain a dose of 4 grams of DHA from 20% oil compositions, it would be necessary to take 20 grams of oil or 40 conventional soft gels of 500 mg).
Polyunsaturated fatty acids are highly unstable in their free form, so their oral uptake requires stabilisation, which can be achieved through esterification or link to other molecules such as glycerol and ethanol, that makes them more stable and increases their bioavailability. The first vehicle (glycerol) enables a higher purity (triglyceride), stability and exhibits a maximum bioavailability, avoiding the presence of alcohol, which is particularly important in cases where high doses are required and when, in numerous applications, its use is chronic, in addition to pregnant women and children. Triglycerides are ingested in concentrations hundreds of times higher as part of the normal food and are the natural nutritional source and pharmacokinetics of fatty acids in target tissues. The pharmacokinetics of triglycerides is maximum and it enables the maximum purity of the active substance, DHA, and therefore DHA triglycerides exhibit the best absorption and have more physiological and metabolic implications.
Parallel to an increased sensitivity towards the connection between food and health, there is an increased acceptance of fish as a healthy nutritional source. Fish is an important source of high quality proteins, minerals and vitamins, in addition to polyunsaturated omega 3 fatty acids, whose benefits for the health are well recognised. However, a recent study highlights the risk associated to environmental contaminants such as mercury and dioxins, which are known to accumulate in fish.
The synthesis of fatty acids and the trophic chain in living organisms are responsible for the physicochemical properties of the membranes and their physiological adaptation to the environmental conditions (ie. temperature). The fatty acids with the lowest fusion point (FP) found in phospholipids and fats of living organisms are pristanic acid (PA), phytanic acid (PhA), EPA, DHA, arachidonic acid (AA) and estearic acid (SDA), which grant optimal physiological conditions under low temperatures. Organisms use two chemical strategies to obtain fatty acids with a very low FP: methylation and the unsaturation of carboxylic acids. Taking into consideration the extreme conditions encountered in organisms of marine origin, it is not surprising that the most rich sources of such fatty acids are mainly in cold marine waters. In this sense, the fatty acids with the lowest FP encountered in nature, foods and in the fatty derived products such as oils of marine origin, are the long branch chain fatty acids (C>18) with several methyl groups, from which PhA is the one present in the highest concentrations and the long chain lineal omega 3 fatty acids (C≧18), where the two most relevant ones due to their concentration and abundance are DHA and EPA.
PhA is present in the human diet or in animal tissues where it can get through the chlorophyll of plants. PhA is formed from the corresponding alcohol, phytol and is oxidised to form PA, which explains why it is usual to find PA and PhA together. PhA is involved in one human pathology, Refsums Syndrome, which is characterised by an accumulation of PhA in the blood and tissues, having discovered subsequently that it is connected with a deficiency in the alpha-oxidation route in the liver.
Whilst the majority of foods contain less than 5 μg of PhA/g, those with the highest content exceed 1 mg of PhA/g, and in the case of fish they exceed 750 μg/g, being its quantity proportional to the percentage of fat. It is considered as the food with the highest concentration of PhA and of higher risk (Group III) for consumption in diseases such as Retinitis Pigmentosa (RP) and defects in the oxidation of PhA such as Refsum's disease. On the other hand, processes for the separation of fats in the production of fish oil, significantly increase the concentration of PhA.
It is known that the fatty fraction from fish is the main source of the omega 3 fatty acids DHA and EPA in the diet, but it is also the main source of PhA. Sources with the highest content in EPA and DHA represent the majority of the intake of DHA and EPA, being associated at the same time with the highest concentrations and intake source of PhA. Both DHA and PhA share the same sources, far beyond products for food and pharmaceutical use, thus finding the highest concentrations of DHA and PhA in products of marine and microbial origin. Normally, PhA is found together with EPA and DHA. Bacteria, fungus and micro-algae are the organisms in nature with the highest concentration of PhA. Oils derived from micro-organisms with a rich content in DHA and the least proportion of PhA, frequently exceed 100 μg/g.
All the western diets studied, including the Mediterranean one, have a daily intake of 100-150 mg of DHA, whilst numerous authors establish a daily need of 200-300 mg. DHA is the only nutrient for which practically all the population worldwide is deficient in their diet.
A very important point which is not usually shown in the literature is that DHA from the diet is 100% of animal origin, since there is no DHA in vegetal food sources (except for some algae which are not used as foods). Therefore, the need in vegetarians is much higher, particularly in strict vegans, where the lowest levels can be found. Its deficiency is even greater and considered by some authors as a marker, in the disease Retinitis Pigmentosa (RP) as well as in metabolic neurodegenerative diseases associated with peroxisomal defects.
Given the widespread interest in the purification of omega 3 fatty acids, mainly DHA, EPA or both, for decades have been numerous patents and regular procedures to obtain refined oils rich in DHA and EPA to obtain higher purities.
However, as shown in FIG. 3, refined and purified oils which exist in the market and that are obtained with patented processes and Good Manufacturing Practice (GMP), contain high levels of PhA, even in those oils with a high DHA purity.
Negative Effects of PhA in Health.
PhA is a risk factor for public health since it induces cancer in prostate, breast, colon . . . as well as neurological and visual disorders (Lloyd-M D et al., 2008; Allen-N E et al., 2008; Thornburg-G et al., 2006; Xu-J et al., 2005). In addition, it is cytotoxic (Komen-J C et al., 2007; Schönfeld et Reiser, 2006; Schönfeld et al., 2006, Heinz, 2005; Elmazar & Nau, 2004). The intake of PhA is a risk factor for the development and/or evolution of diseases: ophthalmologic (retina, cataracts, dry eye . . . ), olphative and auditive alterations, neurologic (Alzheimer, encephalopathy . . . ) and psychiatric alterations, nephrologic alterations, cardiovascular (electrical abnormalities S. Purkinje, alterations of the smooth muscle, ischaemic cardiopathy, atherosclerosis), myopathies and severe amiotrophy, bone alterations, hepatic alterations, alterations of the male and female fertility, chronic autoimmune and inflammatory diseases (Crohn disease, Colitis ulcerosa, LES) and cancer (prostate, colon, breast, kidney, ovary, some types of leukaemia, etc.).
The exact mechanisms by which PhA is toxic for neurosensorial and neural tissues, heart, kidney, liver, intestine, smooth and striated muscle, prostate, breast, sperm, lung and bone system is being gradually elucidated. The most well known mechanisms are connected with the over-expression of tumoral markers (the alpha-methylacil-CoA racemase (AMACAR) or the SPC-2) and the uncoupling protonophoric action of the respiratory electron transport chain in the mitochondria and in the cytoplasmic membranes (ie. phototransducción in retina). At a very low dose, PhA is one of the molecules with the highest induction of oxidative stress in vivo, it potentiates the theratogenesis and is a great inductor of atherosclerosis and death by cardiac failure.
PhA is directly toxic to the mitochondria and exhibits a powerful atherogenic activity. PhA has a rotenon type activity in the uncoupling of complex I in the oxidative phosphorylation in the inner mitochondrial membrane, resulting in the subsequent production of oxygen reactive species and the in vivo lipoperoxidation of DHA and other polyunsaturated fatty acids or PUFA (Kahler-S et al., 2005). It reduces the levels of DHA in phospholipids, mainly in the photoreceptors and neural tissue, increasing the sensibility to ischaemia, to cardiovascular reperfusion lesions and to the oxidation of low density lipoproteins (LDLox), increasing the macrophages anti-inflammatory activity, reducing the energetic and metabolic activity (inhibiting the oxidative phosphorylation) and inducing the mutation of the mitochondrial DNA. This toxic metabolic activity explains by itself why photoreceptors, pigmentary epithelium, neural tissue, heart (Purkinje cells), kidney, liver, ovary, sperm, lungs . . . all of the tissues rich in mitochondria, are the first ones in being affected in patients with high PhA concentrations. PhA induces Ca+2 mediated apoptosis in the Purkinje cells (Powers-J. M et Al. 1999) and sudden cardiac death in animal models. PhA produces ischaemia, apoptosis of the vascular smooth muscle, is atherogenic and particularly cardiotoxic. A deficiency in the sterol-2 transport protein (SPC-2) produces sudden cardiac death by accumulation of PhA in mice. PhA induces apoptosis in cell cultures of vascular smooth muscle cells (VSMC) in humans, mice and pigs.
The altered activity of Ca+2 reabsorbtion and the apoptosis of osteoclasts due to PhA results in bone abnormalities. Proteins linked to Ca2+ in the membranes of the outer segments of photoreceptors, where calmoduline is particularly concentrated, are responsible for the Ca2+ flow which controls multiples event in photoreceptors, including phototransduction and synaptic transduction. The calmoduline function is mediated by numerous proteins linked to it including GTPases. When the calmoduline concentration is reduced in photoreceptors, defects occur in the vision, particularly in the adaptation to light and darkness.
PhA is directly toxic to the cilliary ganglionar cells affecting parasympatic ganglionar nerves in the posterior region of the ocular orbit, responsible for pupil contraction and for the vision accommodation (presbyopia, hypermetropy, photosensibiity, etc). PhA interferes with the function of cilliary cells, basal body and proteins linked to microtubules required for the biogenesis of the cillium, mediated by the interaction of the different types of myosin and prenilation of Rab GTPases in the primary cillium of photoreceptors, affecting the transport of essential proteins such as opsine, olphatory cells, cochlea, renal cells, respiratory system, sperm, intestinal microvilli, as well as to the movement of melanosomes in the pigmentary epithelium in retina. RP, which exhibits alterations in the metabolism of PhA, is a model of the alteration of cilliary cells, finding abnormalities of the sperm axonema and of the rods cilliary cells, affecting to the renewal of the outer segments of the rods which depend on the cilliary body, resulting in irreversible visual damage. Also, in RP with non associated syndromes, alterations of the auditive evocated potentials and alterations of the audiometry compatible with cochlear alterations characteristic of cilliary cells are found. Significative alterations of the cilliary body result in RP and deafness in a way which is comparable to Usher and other syndromes such as in patients non associated to syndromes with auditive alterations.
The Rho and Rab family small G-proteins require addition of these isoprenyl moieties at their C termini for normal GTPase function. Rho GTPase signalling pathway are critically needed to target for therapeutic intervention in nephrological diseases, neurological disorders (myelinisation), cancer progression, cardiovascular diseases, infectious diseases, etc. PhA and other isoprenoids impaired Rho-GTPase signalling, particularly Rac pathway, in an opposite way to DHA and statins. The PhA alters Rho-GTPases in a way comparable to some bacterial toxins in the epithelium and digestive and respiratory mucosa to develop the invasive and infectious processes; tumoral processes and metastasis (ie. prostate, breast), renal lesions (glomerular, tubular etc.) and demielinisation.
These GTPases are the main mechanism that explains why DHA without PhA is more efficient than the rest of DHA with PhA in the numerous applications of in the present innovation.
Following this same line, PhA and PA are controllers of the main and most powerful mediators of the angiogenesis and inflammation phenotypes. The induction of angiogenesis, TNFalpha, GBP-1, GBP-2 and inflammatory cytokines (CI) by PhA and PA is a determining factor for the development of cancer (metastasis), autoimmune diseases, inflammatory diseases, infectious diseases, renal, pulmonary and neurological.
PhA becomes PA by oxidation and both PhA and PA, induce apoptosis mediated by the formation of UNAM of the nitric oxide synthase and high concentrations of the protein within 2 hours from the treatment (Idel et al., 2002). Besides, PhA and PA control the main and most powerful mediators of the angiogenesis phenotype and the inflammation. Also, PhA and PA are the most powerful inducers of the activation and secretion of the tumor necrosis factor α (TNFα) (Idel et al., 2002). The expression of the human guanylate-binding protein (GBP)-1 is highly induced by inflammatory cytokines (ICs) and therefore, may characterise IC-activated cells. GBP-1 is a novel cellular activation marker that characterises the IC-activated phenotype of endothelial cells. GBP-1 is a major regulator of the anti-angiogenic response of endothelial cells to ICs. GBP-1 is a cytoplasmic protein and its expression in endothelial cells is selectively induced by interferon-gamma, interleukin-1alpha, interleukin-1beta, or TNF-alpha, but not by other cytokines, chemokines, or growth factors. PhA and PA are inducers of alpha TNF and of interferon gamma. GBP-1 expression is highly associated with vascular endothelial cells but was undetectable in the skin, but it was highly induced in vessels of skin diseases with a high-inflammatory component including psoriasis, adverse drug reactions, and Kaposi's sarcoma. It has been shown that the expression of GBP-1 and of the matrix metalloproteinase-1 (MMP-1) is inversely related in vitro and in vivo, and that GBP-1 selectively inhibits the expression of MMP-1 in endothelial cells, but not the expression of other proteases. The latter finding indicated that the inhibition of capillary formation is specifically due to the repression of MMP-1 expression by GBP-1, and is not affected by the anti-proliferative activity of the helical domain of GBP-1 (Guenzi et al., 2003).
PhA potentiates the theratogenic effects of retinoic acid (Elmazar & Nau, 2004), being particularly relevant, since DHA, which is frequently associated with PhA, is recommended during pregnancy, nursing and in children food.
Retinosis pigmentaria (RP) is an ideal model to study the toxicity of PhA in presence of DHA, as will be seen further along this document. The physicochemical properties of PhA makes it into an important competitor of DHA when becoming incorporated into the position 2 of phospholipids (the usual position of DHA in photoreceptors). PhA has a high number of free rotating bonds (14) and has a very low crystallisation point, enabling a high fluidity in the membrane. However, PhA is lacking the structural conformation characteristic to DHA for the Van der Waals interactions, with the alpha-helix of rhodopsin, necessary for its mobility in the membrane and the tertiary structure. As a consequence, PhA reduces the activity of rhodopsin and of the phototransduction. PhA has the ability to uncouple the photoreceptors membranes resulting in a failure in the phototransduction (continuous hyperpolarisation) found in RP. PhA is not sensitive to the degradation by oxidation and is resistant to dystrophic conditions such as those found in RP. This happens especially in cases of DHA deficiency, such as in RP, consequence of the dystrophy, where it is the single disease where there is a deficiency in DHA, being considered as a marker of the disease.
PhA modifies the function of photoreceptors through its incorporation to the phospholipids and triglycerides, displacing DHA (McColl & Converse, 1995; Powers et al., 1999, Mönning et al., 2004) and reducing the levels of DHA due to the lipoperoxidation and mitochondrial damage induced by PhA. PhA also has the toxic ability to act as a protons transporter (uncoupling agent) not only in mitochondria but in the photoreceptors membranes (Gutknecht-J, 1988), thus altering the polarisation of the outer segments of the photoreceptors.
Displacement of DHA by PhA.
PhA modifies the function of the photoreceptors through its incorporation into the phospholipids and triglycerides, thus displacing DHA from the second carbon of the phospholipids in the ROS membranes and the mitochondria. Therefore, the displacement of DHA from the membranes by PhA alters the function of the photoreceptors, behaving as a DHA antagonist in the phototransduction, altering the calcium homeostasis and the regeneration of rhodopsin. The displacement of DHA by PhA is one of the various mechanisms of action in some diseases (ie. RD=Refsum's Disease) being a partially pathogenic mechanism.
Whilst DHA is an inhibitor of apoptosis as well as a neurotrophic or survival factor for photoreceptors, PhA is one of the most powerful inductors of oxidation and apoptosis in vivo, interfering on an antagonist manner with the action mechanisms of DHA. PhA levels in the oil interfere with the activity of DHA, since capacity of DHA to inhibit apoptosis in retina is reduced by the presence of PhA on a dose dependent manner.
PhA produces apoptosis in photoreceptors in animal models with RP producing an irreversible damage in mitochondria. Under these conditions, the peptides responsible for the survival of photoreceptors are incapable of inhibiting apoptosis. However, DHA is the single fatty acid that neutralises the reactive species in retina under dystrophic conditions. PhA is particularly toxic in dystrophic retinas and DHA reduces its effects. It has been proved that DHA is an inhibitor of oxidative stress and the irreversible mitochondrial damage which causes photoreceptors degeneration (Rotstein et al. 2003).
Therefore, it can be concluded that PhA besides being cytotoxic, is an antagonist of DHA with all the implications involved. Specifically, PhA on an antagonist manner to DHA, induces apoptosis via mitochondria.
In the current invention two preclinical experiments have been developed inducing apoptosis in photoreceptors with Paraquat (FIG. 1) and MNU (FIG. 2), in order to study the anti-apoptosis effect in vitro and in vivo of DHA in connection with the PhA concentration. In both experiments it is demonstrated that the anti-apoptosis capacity of DHA in photoreceptors is inversely correlated to the PhA concentration. The highest anti-apoptosis activity is obtained with concentrations between 0 and 20 μg/g here the ratio Bcl-2/Bax was significantly lower than in mice fed with <5 μg/g.
Toxicity of PhA.
It is not necessary a metabolic alteration or pharmacologic or alimentary interaction of the oxidation of PhA in order to exhibit toxicity, since a nutritional dose of PhA through a conventional diet, also results in significant variations of PhA and an increased risk for health and toxicity. On an schematic way, the toxicity of PhA through its consumption at low doses, is related with the following situations:    1. Within the oxidative process of PhA is included part of the physio-pathological process: the induction of AMACAR. The effect of PhA in health is also determined by the over-expression of certain molecular markers associated to numerous cancers of great epidemiological value: prostate cancer, colorectal cancer and of renal cells. There is evidence that this same marker is connected with ovary, breast and endocrine cancers related with a resistance to insulin. PhA is essential for the survival of several tumor cell lines (prostate, kidney, breast, colon, lung) resulting in the over-expression of AMACAR and SPC-2. Cases have been described with deficiencies in AMACAR with neuropathy, RP and an increase in PhA and PA (Ferdinanduse et al., 2000).    2. Failure in the oxidation of PhA with an increased accumulation of PhA in patients with RP such as in juvenile or adult Refsum's disease, Zellweger syndrome, neonatal adrenoleukodystrophy and rhizomelic punctata chondrodysplasia.    3. However, other genetic diseases exhibit increases in PhA compared to the normal population due to peroxisomal defects such as in: a) Diseases with mitochondriopathies (Complex IV), COX deficiency, Leigh syndrome, Renal Fanconi syndrome (Fingerhut R et al., 1994).    4. More than 50 mg/day of PhA are eliminated via oxidation and cytochrome P450. It is well known that the oxidation of PhA is mediated by cytochrome p450, but it is also known that that there are strong inhibitors of cytochrome P450 (ie. antimicotic azolics, valproic acid, cimetidine, erithromicine, Sulfametoxazol, opioids, cyclosporine, protease inhibitors, antidepressants, hyperphorine, barbiturics, antihistaminics, tamoxifen, cannabioids, S-warfarine, etc.). Thus, certain drugs can increase PhA concentrations on a significant manner. Numerous pharmacological treatments inhibit the metabolic degradation of PhA, accumulating and becoming mainly retinotoxic, neuro- and cardio-toxic. PhA can interact with calcium antagonists, antiangiogenic agents, immunosuppressors and antiinflammatories. The fast oxidation of PhA (alpha-oxidation=90% oxidation of PhA) in the human body is mediated by different isoenzymes of cytochrome P450 (ie. CYP2C8). One the most outstanding drugs, due to its commercial interest and use are the hypolipemiant drugs: fibrates and statins, inhibitors of P450 CYP2C8 which strongly inhibit the oxidative metabolism of PhA, resulting in an accumulation of PhA. Fibrates activate the β-oxidation in peroxisomes, but the degradation of PhA needs the alpha-oxidation which is inhibited by fibrates through the inhibition of cytochrome P450 CYP2C8. PhA is likely to be partly responsible for its main secondary effect: rhabdomyolysis.    5. Secondary failures in the oxidation of PhA: in Alzheimer's disease the activity of the thiamine-dependent enzymes in peroxisomes is reduced, resulting in a reduced amount of the hydroxyphytanoyl-CoA lyase necessary for the oxidation of PhA. Parallel to the increase in the acethylcholine levels found in Alzheimer's disease, an increase in PhA levels has been found.    6. Nutritional alterations (antimetabolites: thiaminases and thiamin antagonists, acetylcholine) that affect the decrease in thiamin, where thiamin pyrophosphate is a cofactor in the oxidation of PhA (via ligases), as well as thiamin deficiencies from which the most well known are those related to the Wernicke-Korsakoff syndrome, the fatal cardiovascular disease beriberi and the neurotoxic syndrome due to consumption of carp and salmonids. Thiamin antagonists are found in substances such as food preservatives (ie. sulphites) from plants and frequent foods (ie. tea, grapes, citrics . . . ), resistant to boiling (ortho- and hydroxiphenols) such as caffeic acid, chlorogenic acid and tannic acid, quercetine and rutine (very used in pharmacology and as food supplements); thiaminases from foods (frequent in fish, mainly from fish farms, 80% of consumption), rumen, diary products and ruminants meat: foods rich in PhA); alcohol consumption, grapefruit juice (and to a lesser extent orange juice, mandarine, apple, grape and their derivates) and caffeine can increase to a lesser extent the deposits of PhA. An interesting model is a genetic disease that produces deficiency of thiamine and neuro-sensorial deafness. Alcohol and pyrithiamin (thiamin antimetabolite) only require 100 μg/ml in order to produce a severe deficiency of thiamin in less than 7 days. These interactions bring to the attention that it is not only important to assess the amount of PhA and phytol contained in food, but that foods associated to the diet, pharmacological treatments, food supplements and habits, can affect the accumulation of PhA from the diet.    7. On the other hand, epidemiological data leave no doubts regarding the toxic effect at doses which are considered normal (50-100 mg/day), being toxic at doses as low as 0.1 μmol/mg fat. Even at much lower concentrations, PhA is one of the molecules with the strongest capacity to induce oxidation in vivo, interacting with some essential physiological mechanisms of DHA and reducing its levels in vivo, damaging its structure through lipoperoxidation. At very low blood concentrations 300 μg/ml or <1 mmol/l (<1% total fatty aids) and approx. 5-10% of the total fatty acids of the nervous tissue, it creates a severe neuropathy and death. In post-mortem toxicity studies associated to the accumulation of PhA (Refsum disease), the highest levels found in just some tissues had reached 8.5% of the total fatty acids23. Refsum's disease both in adults and children with a severe accumulation of PhA, is an ideal model to study the toxicity by PhA which includes retinosis pigmentosa, nistagmus, hypotony, ataxy, mental and growth retardation, facial and bone dysmorphies, hepatomegaly and hypocholesterolemia.DHA and PhA
PhA toxicity is related with diseases and situations where the intake of DHA is recommended and used, such as in retinosis pigmentosa (RP), where DHA behaves as a marker of the disease and PhA is the etiological agent of RP. Diseases which commonly require DHA for their treatment, are induced at the same time by PhA. Besides the oncologic ones, the most evident one of all is RP. The branch chain fatty acids PhA and PA, are markers in different RP diseases (Refsum, Neonatal Adrenoleukodistrophy (NALD), Chondrodysplasia punctata rhizomelic, Zellweger, Usher IV) associated with a defect in the metabolism of the alpha- and beta-oxidation of PA. PhA is the single cause of RP in these cases.
For decades, it has been known that in RP there are deficiencies in DHA in all target tissues. All patients with RP exhibit alterations in the metabolism of DHA and a significant part of them have high concentrations of PhA which, to a certain extent, is the cause of the disease evolution. The degree of DHA deficiency is not associated with the evolution and prognosis of the disease. In this sense, the non systemic dominant autosomic RP is the most benign of all the hereditary forms (PhD Thesis Cela-López, J M), even at DHA levels which where below those taken on an sporadic basis (Schaefer et al. 1995). However, the oral intake of 2 g/day of DHA in patients with XLRP does not normalise the levels of DHA, due to a loss of DHA in dystrophic retinas, being necessary to take 4 g/day in order to normalise the levels of DHA in the erythrocytes phospholipids. The evolution and prognosis of the various types of RP, depends on the pharmacological dose of DHA and of the moment of starting its intake, as well as of the PhA levels. Therefore, it can be said that the concentration of PhA is related with the worse prognosis in the evolution of the disease and in the loss of the central function (macular region).
Increased Levels of Phytanic Acid have been Found in Treatments with “DHA”
Four samples from 4 patients arrived to our laboratory to evaluate the beneficial effect of a treatment with DHA in two syndromes with RP. Treatment with 4 g/day of DHA (4.86 mg phytanic) of four patients with juvenile Refsum disease and Zellweger syndrome during 3 months, increased the presence of branch chain fatty acids: phytanic and pristanic by 50% and 44%. On a parallel way, a complete worsening of the clinical condition was observed: neurological, deafness and RP (visual acuity and visual field). The Peroxisomal Disease Laboratory from the Kennedy Krieger Institute (Baltimore), routinely informs in all the analytical results to all the patients that they study during the year, that intake of DHA from fish increases the toxic levels of phytanic acid and therefore they always recommend DHA from algae. Data show that commercial concentrates of DHA are toxic in patients with RP associated to peroxisomal defects.
In 1994, the Association of RP Patients (AARPE Spain) carried out a study with 17 patients with RP with or without syndromes (Refsum, Zellweger, NADL, Kearns, Bordet-Bield, autosomic recessive RP, and sporadic RP) that had increased levels of phytanic acid in plasma. They all took DHA oil with different amounts of phytanic acid (from 5 mg to 11.5 mg per day) in variable periods of 1 month up to 3 years. They all showed increased levels of phytanic acid from 23% up to 82%, regardless of the fish oil source. After 1 year, the progression of RP was greater than expected in RP patients with non syndromes (11 patients) (VA 8.3% less), although in those with syndromes (6 patients) the loss in visual function was quite valuable. Subsequently, the treatment with DHA rich in phytanic acid was removed and patients were separated into two groups according to plasmatic levels of phytanic acid (moderated levels: 5-30 μg/ml; high levels 30-900 μg/ml). Treatment with 4 g of DHA low in phytanic acid (<90 μg/ml) was introduced and the levels of phytanic acid and the visual function were assessed. In the group with moderate phytanic levels (no associated to syndromes), a progressive reduction of phytanic acid levels was observed which became normal within 12 months (FIG. 4). Parallel, a regression in the progress of the disease was observed with a visual function comparable to that obtained before the beginning of the treatment with DHA rich in phytanic acid.
The Toxicity of PhA is Linked with the Consumption of Sources Rich in DHA and EPA (ie. Tunna) Since it is Present in Oils at Very Low Percentages (Approximately 0.1%).
PhA is found in the human diet or in animal tissues where it can derive from chlorophyll from plant extracts; this is how it can accumulate in animal tissues. PhA is formed from phytol and is oxidised forming pristanic acid (PA). Given the important variations that exist in the fatty diet of the population (vegetarians, ovolactovegetarians, . . . ), variations in PhA blood levels have been found (up to 6.7 times) exclusively related with the consumption of PhA from the conventional diet, where vegetarian diets exhibit up to 10 times less PhA whilst at the same time are extremely deficient in DHA.
Whilst phytanic acid is a risk factor for prostate cancer, DHA is a protective factor for the same type of cancer. Evidence exceeds to the numerous molecular studies and is supported by epidemiological data and numerous pharmacologic studies, some of which are well advanced (Phase II), regarding the role of DHA in combination with other chemotherapeutical drugs (celecoxib, Plaquitaxel) in the prevention and first line treatment of prostate cancer as well as gastric cancer (Ballet et al., 2004; Jones et al., 2007). Also there is sufficient epidemiologic evidence of the role of phytanic acid in the induction of prostate cancer (Walsh, 2005; Xu et al., 2005; Thornburg et al., 2006; Mobley et al., 2003). In addition, there are three separate studies which associate the take up of fatty fish, red meat and dairy products (sources with the highest concentrations of phytanic acid) with prostate cancer.
In the patents field, it is known of documents to purify oils that contain EPA and DHA, such as document U.S. Pat. No. 4,874,629 (1989) which refers to a procedure to treat oils that contain omega 3 fatty acids, such as salmon, pilchards and other fish which contain EPA and DHA and that in essence consists of: a) subject the oil to a vacuum distillation at 30-150° C. during 2-5 hours and putting in contact the oil with an adsorbent selected amongst silica gel and silicic acid to reduce the high boiling temperature and the most volatile polar flavours and other non desired constituents such as polymers, cholesterol, pigments, pesticides ad heavy metals; and b) subsequently recover the oil from the mixture. Later on, the same authors in U.S. Pat. No. 5,023,100 apply the previous procedure in order to produce an edible oil with EPA and DHA, that can be combined with vegetal oil and/or rosemary oil to improve its oxidative stability.
Likewise, document US2008/0268117 A1 describes a method to purify oils that contain EPA and DHA which comprises: (a) add to the oil an aliphatic alcohol of C1-C4, preferably ethanol in an aqueous solution at 60-70%, at a temperature to which the oil and alcohol separate into two phases (round about 10° C.); (b) heating the mixture until the oil and the alcohol become miscible (50-80° C.): (c) cooling down the mixture at a temperature in which the oil and alcohol separate (approx. 10° C.); and (d) recovery of the oil phase. It is specified that such process is especially adequate to prepare oils to be used in foods and pharmaceutical products due to the fact that the aforementioned process, eliminates the organic contaminants such as cholesterol and heavy metals such as mercury. In facts, it mentions that oils prepared on such a way, are especially suitable to prepare an infant formula (U.S. Pat. No. 5,013,569) and compositions for the treatment of rheumatoid arthritis (U.S. Pat. No. 4,843,095).
There are also documents for obtaining EPA and DHA triglycerides, as for example, document ES 2035751 T3 which refers to a procedure to prepare a triglyceride which has at least one long fatty acid C8+ in the molecule, characterised by an interesterification, in the presence of a lipase, the free long chain fatty acid or one of its inferior alkyl esters C1-C4 with a triglyceride which has one or more short chain fatty acids C2-C6 in the molecule and separate by evaporation during the reaction, the short chain free fatty acid or its inferior alkyl ester, and composition in which the polyunsaturated acid is EPA or DHA or a mixture of both. Document GB 2350610 A describes the preparation of DHA from this oil as a triglyceride through a procedure that uses a combination of a transesterification of the triglycerides with an inferior alkylic alcohol, distillation and a selective enzymatic transesterification with an alcoxy alcohol catalysed by lipases that can be immobilised. Also, document US2008/0114181 A1 refers to a method for the esterification of fatty acids into triglycerides with aliphatic alcohols C1-C8. The method uses an acidic ion exchange resin as a catalyser, which comes into contact with the mixture of the reaction which contains a triglyceride that at least has 1% of free fatty acids and an aliphatic alcohol C1-C8, in adequate conditions for the esterification.
Likewise, it is known how to obtain EPA and DHA as ethyl esters, for example in document U.S. Pat. No. 5,679,809, which describes a procedure for obtaining a concentrate of ethyl esters from polyunsaturated fatty acids, preferably EPA and DHA, which consists of mixing the oil which contains the fatty acids with ethanol in the presence of a catalyser to form an ethylic ester of the fatty acid, whose phase is separated mixing it with urea and ethanol, which is then cooled down until a solid phase is created and then, the liquid phase is separated from which a fraction is obtained enriched with the desired polyunsaturated acids. Another document which also uses urea to separate saturated fatty acids and the majority of the mono-unsaturated ones from the rest of fatty acids present in marine animal oils is EP 0347509 A1, which obtains as a final product a mixture of EPA and DHA. Document U.S. Pat. No. 5,734,071 which achieves a product which contains EPA+DHA from a fish oil using a similar method with urea and the ES 2018384 which prepares a composition with EPA and DHA in relative amounts of 1:2 to 2:1, where these fatty acids constitute 75% in weight of the total fatty acids, also with a method that uses concentration by fractioning with urea and molecular distillation and/or extraction with fluid in super-critic conditions or chromatography.
Document ES 2056852 T3 also claims a procedure for the extraction of the ethylic ester of DHA from fish oil, which includes the transesterification of the fish oil with ethanol in presence of sulphuric acid, followed by the extraction of the mixture with hexane, silica gel chromatography, treatment of the residue in acetone cooled down to −40° C., filtration, evaporation of the acetone and molecular distillation in two steps at 0.133 Pa, the first step at 80-100° C. and the second at 105-125° C.
There are a large number of documents that mention the use of DHA for various states of need and diseases previously mentioned. As an example, we will mention document CN 1557453 (A) which describes a composition to increase memory and improve knowledge which comprises the serrate herb clubmoss, rhodiola root and a fish oil concentrate with 50 mg de DHA as an active substance. It specifies that it strengthens the transmission of the information between neuronal synaptic connections, improving the resistance to anoxia, stabilising the structure of the nervous cells and supplying such nerve cells with essential nutritive material.
In addition, DHA has been mentioned in different documents as having beneficial properties for health, for example, as a nutritive substance for the human brain and to revitalise the intelligence (CN 1130040 (A)). In JP 8098659 (A) it is mentioned that DHA, as an ester or phospholipid, has an improved effect against stress. CN 1105205 (A) gives a description of a capsule which contains 11-45% of pure DHA together with calcium, vitamins and starch, to tonify the brain and activate the intelligence. And document ES 2277557 A1 refers to the use of DHA for the manufacture of a pharmaceutical composition geared towards the treatment of the oxidative cellular damage.
In the state of the art, documents can be found to selectively separate and purify EPA and DHA. Thus, document EP 1065196 A1 refers to a process to selectively separate and purify EPA or DHA or their esters from a mixture of acids or esters which comprises: (a) passing an aqueous fluid with a silver salt through a column with diatom earth, so that the silver salt adheres to the diatom earth; (b) pass through the column with diatom earth and silver salt, a solution with solvents of a mixture containing the fatty acids highly unsaturated or its derivates; and (c) passing a solvent to separate the desired fatty acids.
Document U.S. Pat. No. 6,846,942 B2 refers to a method for the preparation of pure EPA and DHA which comprises: a) dissolving the mixture of DHA and EPA in acetone and adding magnesium ions, which produces EPA and DHA salts which have different solubility in acetone, b) cool down the solution obtained in (a) to precipitate the EPA salt, c) filter the precipitated EPA salt, and d) acidify the precipitate obtained to obtain pure EPA, and e) evaporate the solvent from the filtrate to obtain pure DHA. When the EPA and DHA mixture is obtained from a fish oil, this is firstly subjected to an alcoholysis or saponification in order to transform the triglycerides into free acids.
Therefore, there is still a need for a composition that should not only be beneficial for the diet and health due to the presence of DHA in effective amounts for health, that is, in high amounts of DHA, but at the same time it should contain the least possible amount of PhA in order to prevent its effects when taking in DHA.